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====<u> Ligation reaction Mix</u> ==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:70px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:70px;background:#f0f0f0;"|'''26x rxn''' |- | dA-tailed DNA||25.00||0.00 |- | 10X Tango buffer||0.50||13.00 |- | HC T4 DNA ligase (30units/ul)||1.00||26.00 |- | 10mM ATP ||1.50||39.00 |- | H2O||1.00||26.00 |- | Total||29.00|| |} :- Aliquot 13ul of ligation reaction mix to 8-tube strip <u>PC-P samples</u> :{| {{table}} class = wikitable | align="center" style="width:80px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Index''' |- | PC_P_1||1 |- | PC_P_2||2 |- | PC_P_3||4 |- | PC_P_4||5 |- | PC_P_5||6 |- | PC_P_6||7 |- | PC_P_7||8 |- | PC_P_8||10 |- | PC_P_9||11 |- | PC_P_10||12 |- | NTC_1||11 |- | NTC_2||12 |} <u>NC-P samples</u> :{| {{table}} class = wikitable | align="center" style="width:80px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Index''' |- | NC-1||13 |- | NC-2||14 |- | NC-3||15 |- | NC-5 ||16 |- | NC-6 ||27 |- | NC-7 ||18 |- | NC-8 ||19 |- | NC-9 ||20 |- | NC-10 ||21 |- | NC-11 ||22 |- | NTC_1||21 |- | NTC_2||22 |} :- Add 1ul of 1:20 diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction :- Add 4ul of ligation reaction mix :- Spin down the tube :- Mix by gentle pulse-vortexing on PCR rack 10x :- Spin down the tube :- 16C, 30h (no heat lid) --> I had no time to come back to continue to next step. I incubate longer. :- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on) :- Continue to bisulfite conversion
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